Project description:To investigate the transcriptional remodelling during EMT, we treated normal murine mammary gland epithelial cells with TGFbeta for 0, 2h, 6h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 168h and 240h. Using WGCNA and pathway enrichment analysis we identified multiple gene expression modules that were enriched in general, signaling, metabolic or stuctural pathways highly relevant for EMT.
Project description:To investigate the context-dependent function of Irf1 in maintaining epithelial identity while enabling TGFbeta-induced EMT in NMuMG/E9 cells, we performed chromatin immunoprecipitation with Irf1-specific antibodies in NMuMG cells treated for 2 days with TGFbeta or left untreated (0d TGFbeta). Intersection with RNA-sequencing after downregulation of Irf1 with or without treatment with TGFbeta for two days revealed genes that are directly regulated by Irf1 and that could contribute to the dual role of Irf1 in EMT.
Project description:Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. However, several of the studies have been carried out using model systems that don’t appropriately recapitulate all stages of the dynamic process of EMT. Hence, there is a need to overcome this limitation by development of a model system that allows us to mimic each stage of EMT and accurately assess the plastic changes associated with it. Methods: We have derived a cancer cell line from the PyMT-MMTV model of breast cancer, named PyMT-1099 cells, and undertaken a detailed characterization of the morpho-genetic changes it undergoes during a TGFbeta-induced EMT. Further, we have also performed high throughput transcriptomics on PyMT-1099 cells undergoing EMT in a high resolution kinetic of TGFbeta treatment. Results: We show that PyMT-1099 cells undergo an EMT comparable to the classically used immortalized NMuMG cells as assessed by morphological, and marker expression changes on TGFbeta treatment. Further, PyMT-1099 cells can also migrate in vitro in response to TGFbeta treatment. These cells are also tumorigenic and lead to metastasis formation when transplanted into immunocompromised mice. Conclusion: In this study we report the development of PyMT-1099 cells as an excellent tool to model and study breast cancer-associated EMT both in vitro and in vivo and show that these cells overcome the limitations posed by other cellular systems currently being used to study EMT
Project description:To investigate the context-dependent function of Irf1 in maintaining epithelial identity while enabling TGFbeta-induced EMT in NMuMG/E9 cells, we downregulated Irf1 by siRNA and analyzed differentially regulated genes and pathways upon EMT induction (2 days TGFbeta) or in the absence of EMT (0 day TGFbeta). Intersection with Irf1 ChIP-sequencing after 2 days of TGFbeta treatment or in untreated cells revealed genes that are directly regulated by Irf1 and that could contribute to the dual role of Irf1 in EMT.
Project description:We have identified the transcription factor forkhead box protein F2 (Foxf2) to be upregulated in its expression during the EMT process and studied its functional contribution to EMT by siRNA-mediated knockdown in NMuMG cells treated for 4 days with TGFbeta followed by mRNA-sequencing. Our analysis revealed a dual role of Foxf2 during TGFbeta-induced EMT in promoting apoptosis while inducing cell junction breakdown and migration.
Project description:Although heterochromatin is enriched with repressive traits, it is also actively transcribed, giving rise to large amounts of non-coding RNAs. Although these RNAs are responsible for the formation and maintenance of heterochromatin, little is known about how their transcription is regulated. Here we show that the Snail1 transcription factor represses pericentromeric transcription, acting through the H3K4 deaminase LOXL2. Since Snail1 plays a key role in the epithelial to mesenchymal transition (EMT), we analyzed the regulation of mouse heterochromatin transcription in this process. At the onset of EMT, one of the major structural heterochromatin proteins, HP1a, is transiently released from heterochromatin foci in a Snail1/LOXL2–dependent manner during EMT, concomitantly with a down-regulation of major satellite transcription. Global transcriptome analysis indicated that ectopic expression of heterochromatin transcripts affects the transcription profile of EMT-related genes. Additionally, preventing the down-regulation of major satellite transcripts compromised the migratory and invasive behavior of mesenchymal cells. We propose that Snail1 regulates heterochromatin transcription through the histone-modifying enzyme, LOXL2, thus creating the favorable transcriptional state necessary for completing EMT. Keywords: Expression Profiling by array We analyzed 2 arrays from each condition: Control and Major treated 8 hours with TGFbeta
Project description:We report the gene expression in mouse NMuMG cells with or without UGCG depletion using RNA sequencing. After data analysis, the 11218 transcripts were identified. Among these transcripts, the TGF-β signaling target genes and EMT marker genes were significantly upregulated in UGCG depleted NMuMG cells. Then we used these genes and mouse TGF-β or EMT gene signatures to do the gene set enrichment analysis and showed the negative correlation of UGCG and TGF-β signaling or EMT in NMuMG cells.
Project description:Analysis of NMuMG cells expressing Osr2 or treated with TGF-ß. Results provide insight into a novel function of Osr2 in EMT induction.
Project description:We report the application of single cell RNA-sequencing using indrop on an HMLE breast cancer cell line that we induced to undergo EMT. We measured 7523 single cells after 8 and 10 days of stimulation with TGFbeta. In addition, we measured 3496 single cells in an engineered HMLE cell line with Dox inducible Zeb1, after 2 days of stimulation with Doxycycline. Finally, we performed ATAC-seq on CD44 sorted HMLE cells after 8 days of stimulation with TGFbeta.